Periodic Reporting for period 2 - RHIZOCARBON (Forest belowground carbon transport: From rhizosphere fluxes to physiological drivers) Reporting period: 2021-03-01 to 2022-08-31 Summary of the context and overall objectives of the project To facilitate predictions of carbon storage in changing climates and forests, scientists are looking at the trees. Tree carbon allocation dynamics are important, not only for tree eco-physiology, but also for global biogeochemistry. Despite extensive research in tree aboveground carbon fluxes, little is known about what is happening belowground. The EU-funded RHIZOCARBON project will investigate belowground carbon flow. It will study carbon flow in and between tree roots, including their fungal partners in the forest soil. Computational models will be applied to identify the evolutionary requirements for the development of belowground carbon transfer. To trace belowground carbon transport, the project will apply a new methodology of continuous in vivo combined measurement of 13CO2 carbon allocation and flux rate. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Our proposed rhizosphere research has two major arms: the carbon sink partitioning arm (Objective 1) and the tree-tree carbon transfer arm (Objective 2). To accomplish Objective 1, we will measure all tree carbon fluxes simultaneously and unify them to construct a tree carbon balance (Aim 1.1). The carbon compounds that are being exported from tree roots will be identified (Aim 1.2). Then, we will apply 13CO2 labelling and detection to partition among the different belowground carbon sinks (Aim 1.3). This method uses a colorless tracer CO2 which is almost completely comprised of the rare carbon-13 stable carbon isotope.In the first period of the project, we made progress on each and every aim of objective 1. Tree carbon fluxes were measured simultaneously in our forest site since the start of the project, and the raw data are now being unified to construct a tree carbon balance (Aim 1.1). We also started the identification of carbon compounds that are exported from the root (Aim 1.2) and the manuscript reporting this study has been reviewed and is now under revision.We applied 13CO2 labelling and detection to partition among the different belowground carbon sinks (Aim 1.3) and we have reported the results of this study (Rog et al. 2021). Across five tree species, C moved from leaves to stem and fine roots following an exponential decay (a rapid decrease with time), with parameters that are consistent with the ecological role of each species. Eight days post-labeling, conifers allocated ~30% of the C belowground, and broadleaves <10%. In contrast, the residence time of carbon in leaves was ~4 days in conifers and only ~1 day in broadleaves. Root exudation was quantified and shown to be small. We integrated our results into a simplified tree model of 13C distribution, enhancing our understanding of divergent C allocation strategies among tree species cohabiting within the same ecosystem.To accomplish Objective 2, we will genetically identify the mycorrhizal species populating tree roots and characterize their networks (Aim 2.1). Next, we will apply our isotopic labelling method, this time to trace carbon transfer between trees (Aim 2.2). Finally, we will apply computational modelling to test specific hypotheses about the evolution of mycorrhiza-induced cooperation (Aim 2.3). Mycorrhizal fungi are symbiotic to plant roots, and can form belowground networks between individual trees of the same or different species. Climate manipulations will be used to extend the research beyond the current conditions, facilitating predictions of future changes in tree carbon allocation, particularly among belowground fluxes.In the first period of the project, we made progress on the three aims of objective 2. We identified and mapped the mycorrhizal species colonizing tree roots and characterized their networks (Aim 2.1) in the temperate mixed forest stand where tree-tree carbon transfer was originally identified (Klein et al. 2016). The results of this identification were published (Rog et al. 2020), and showed that among the nearly 1,200 ectomycorrhizal (EMF) root-tips examined, 50%–70% belong to operational taxonomic units (OTUs) that were associated with three or four tree host species, and 90% of all OTUs were associated with at least two tree species. Sporocarp 13C signals indicated that carbon originating from labelled Picea trees was transferred among trees through EMF networks. Interestingly, phylogenetically more closely related tree species exhibited more similar EMF communities and exchanged more carbon. Our results show that belowground carbon transfer is well orchestrated by the evolution of EMFs and tree symbiosis.We next applied our isotopic labelling method to trace carbon transfer between trees (Aim 2.2). The results of this study were also published (Cahanovitc et al. 2022). Pinus halepensis and Quercus calliprinos saplings growing in forest soil were labeled using a 13CO2 labeling system. Repeated samplings were applied during 36 days to trace how 13C was distributed along the tree-fungus-tree pathway. To identify the fungal species active in the transfer, mycorrhizal fine root tips were used for DNA-stable isotope probing (SIP) with 13CO2 followed by sequencing of labeled DNA. Assimilated 13CO2 reached tree roots within four days and was then transferred to various EMF species. C was transferred across all four tree species combinations. While Tomentella ellisii was the primary fungal mediator between pines and oaks, Terfezia pini, Pustularia spp., and Tuber oligospermum controlled C transfer among pines. We demonstrate at a high temporal, quantitative, and taxonomic resolution, that C from EMF host trees moved into EMF and that C was transferred further to neighboring trees of similar and distinct phylogenies.We applied computational modelling to test specific hypotheses about the evolution of mycorrhiza-induced cooperation (Aim 2.3). The results of this data-model fusion study were summarized and are now being submitted for publication. Finally, we started implementing climate manipulations that will be used to inform predictions of future changes in tree carbon allocation, particularly among belowground fluxes. We constructed a rain exclusion manipulation in our Mediterranean mixed forest site and continue the measurements there. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) As detailed above, our progress had both novel methodologies and inter-disciplinary developments. As far as we can tell, our research was the first to use the DNA-SIP methodology for mycorrhizal detection. In addition, our collaboration with the research team of Prof. Lilach Hadany (Tel Aviv University) demonstrates a new and inter-disciplinary development in the study of mycorrhizal network formation.Expected results until the end of the project include the completion of all aims as indicated above. This includes:1. Completion of the tree carbon balance in the field2. Calculation of the tree carbon balance under drought3. Synthesis of belowground carbon transfer among trees and mycorrhizal fungi in a mixed forest.4. Completion of the identification of exported carbon compounds.In addition, we are working towards publication of the manuscripts which have been submitted and are currently under revision.